Project description: We propose to develop a new vaccine delivery platform based on a non-pathogenic E. coli with specific safety features. Compared with other bacteria used for vaccines, the E. coli multi-deletion strain (MDS) has been extensively engineered, removing more than 40 large DNA segments totaling 15% of the genome. The deletions are designed to remove all mobile DNA, all genes that could have any role in pathogenicity, and other segments acquired by horizontal transfer, leaving a "clean", stable genome. This strain will deliver Shiga toxin vaccines directly into host cells. In this process, the vaccine vector gains direct access to cells of the submucosal immune system, where the vaccines molecules encoded in the strain are produced, provoking an immune response. The system is designed to deliver any or several vaccines, with a higher degree of safety than other bacterial systems. E. coli can be orally administered and is stable in storage. The genome can be readily resequenced using new chip technology, to give assurance of a completely defined system. On this platform, a vaccine against the Select Agent Shiga toxin will be built and tested. The toxin is a highly potent ribosome-inhibiting protein related to cholera toxin and ricin. It is produced by the enterohemorrhagic E. coli strain O157:H7 and other diarrheagenic strains. The toxin can cause Hemolytic Uremic Syndrome with fatal complications, by attacking microvascular epithelial cells in the kidney and central nervous system. Aims: 1) Construct MDS vaccine strains that contain a mutant Shiga toxin gene and are capable of invading human cells. 2) Evaluate candidate strains in the murine Shiga toxin protection model. Relevance to Public Health: There is no currently available vaccine or therapy to block the fatal kidney damage that Shiga toxin can cause. It presents a serious health threat to susceptible patients and even to healthy members of the general public if the toxin were to be used as a bioweapon. The clean-genome vaccine platform could provide safe protection from this and many other infectious diseases. [unreadable] [unreadable] [unreadable]